(1) Department of Molecular Biology and Genetics, University of Guelph, N1G 2W1 Guelph, Ontario, Canada
Abstract:
We have established a uniform procedure for the expression and purification of the cyclin-dependent kinases CDK7/CycH/MAT1,
CDK8/CycC and CDK9/CycT1. We attach a His6-tag to one of the subunits of each complex and then co-express it together with the other subunits in Spodoptera frugiperda insect cells. The CDK complexes are subsequently purified by Ni2+-NTA and Mono S chromatography. This approach generates large amounts of active recombinant kinases that are devoid of contaminating
kinase activities. Importantly, the properties of these recombinant kinases are similar to their natural counterparts (Pinhero
et al. 2004, Eur J Biochem 271:1004–14). Our protocol provides a novel systematic approach for the purification of these three
(and possibly other) recombinant CDKs.
Published: August 18, 2004.